ABSTRACT . In particular, the The phenomenon of molecular changes to the lymphatic endothelial cells and the biological role(s) of the lymphatic vasculature in the metastatic cascade of human cancer are not entirely understood syndrome of in-transit melanoma is a fascinating clinical example of disease presentation in search of a pathophysiological basis. In our recent paper, Ligand-directed targeting of lymphatic vessels uncovers mechanistic insights in melanoma metastasis (Christianson et al. PNAS, 2015), we performed ex vivo combinatorial screens using random peptide phage libraries of draining lymphatic channels removed directly from patients during melanoma excision surgeries. We discovered a functional ligand-receptor system by selecting, isolating, and validating a new homophilic protein-protein interaction between malignant melanoma cells and lymphatic endothelial cells. This unique and previously unrecognized finding provides the foundation for the development and optimization of a new platform for ligand-directed imaging of the lymphatic system. Here, we propose to interrogate previously identified peptide ligands that bind to the surface of the lymphatic endothelium during disease progression to develop a novel, ligand-directed, non-invasive in vivo lymphatic imaging platform. Specifically, our goals are to: (1) Use innovative chem- and bio-informatics data mining systems to define the ontology of enriched lymphatic vessel homing peptides, (2) Investigate the binding properties of selected peptide ligands and their corresponding receptors in vitro, and (3) Develop and implement targeted imaging systems to study the lymphatic endothelium. Peptides that home to PPP2R1A, a new powerful lymphatic marker, will be prioritized. Our long-term goal, subsequent to the completion of the work proposed here, is to discover new lymphatic biomarkers associated with disease progression and generate a panel of ligand-based imaging agents that specifically target the surface of the lymphatic endothelium for prognostic and diagnostic translational applications. We anticipate that the newly characterized lymphatic molecular addresses and validated, corresponding ligands and antibodies will be of great value for the development of targeted agents, establishing for the first time, a platform for non-invasive imaging of the lymphatic system, and, in the future, contribute to our understanding of the role by the lymphatic vasculature in human disease.